Production of ethanol by microorganisms provides an alternative energy source to fossil fuels and is therefore an important area of current research. Zymomonas mobilis is a bacterial ethanologen that grows on glucose, fructose, and sucrose, metabolizing these sugars to CO2 and ethanol via the Entner-Douderoff pathway. Though wild type strains cannot use xylose as a carbon source, recombinant strains of Z. mobilis that are able to grow on this sugar have been engineered (U.S. Pat. No. 5,514,583, U.S. Pat. No. 5,712,133, WO 95/28476, Feldmann et al. (1992) Appl Microbiol Biotechnol 38: 354-361, Zhang et al. (1995) Science 267:240-243). Xylose is the major pentose in hydrolyzed lignocellulosic materials, and therefore can provide an abundantly available, low cost carbon substrate for use in fermentation. Z. mobilis has been engineered for expression of four enzymes needed for xylose metabolism: 1) xylose isomerase, which catalyses the conversion of xylose to xylulose; 2) xylulokinase, which phosphorylates xylulose to form xylulose 5-phosphate; 3) transketolase; and 4) transaldolase (U.S. Pat. No. 5,514,583, U.S. Pat. No. 6,566,107; Zhang et al. (1995) Science 267:240-243). Through the combined actions of these four enzymes and the cell's normal metabolic machinery, three molecules of xylose are converted to two molecules of glucose 6-phosphate and one molecule of glyceraldehyde 3-phosphate, which are subsequently converted to ethanol and CO2 on the glucose side of the pathway (see FIG. 1).
Though there has been success in engineering Z. mobilis strains for xylose metabolism, the strains do not grow and produce ethanol as well on xylose as on glucose. One factor that causes poor growth on xylose is the production of xylitol as a by-product of xylose metabolism (Feldmann et al. supra; Kim et al. (2000) Applied and Environmental Microbiology 66:186-193). Xylitol is phosphorylated by xylulose kinase to produce xylitol 5-phosphate, which accumulates in the cell and inhibits bacterial growth. Xylitol synthesis also reduces the yield of ethanol, since xylose-utilizing recombinant strains of Z. mobilis cannot convert xylitol to ethanol. In addition, xylitol is a potent inhibitor of xylose isomerase (Smith et al. (1991) Biochem J. 277:255-261), which catalyzes the first step of xylose utilization in the engineered xylose metabolism pathway. See FIG. 2 for a diagram showing xylitol synthesis and effects.
The physiological pathway and enzymes that are responsible for xylitol synthesis in vivo have not been determined. However, it has been demonstrated that cell-free extracts from wild type Z. mobilis are able to reduce xylose to xylitol when they are supplemented with NADPH (Feldmann et al., supra), and that this reaction is catalyzed by an NADPH-dependent aldose reductase. It has also been shown that Z. mobilis cell-free extracts are able to convert a small amount of xylose to xylitol without NADPH supplementation, and that xylitol production under these conditions increases 3- to 4-fold when purified xylose isomerase is also added to the reaction mixture (Danielson, 2001, University of Colorado Masters Thesis). Since xylose isomerase is able to convert xylose to xylulose, the clear implication of the latter experiment is that the Z. mobilis enzyme glucose-fructose oxidoreductase (GFOR) can use xylose as an electron donor and xylulose as an electron acceptor to generate xylitol as will be discussed in greater detail below. Thus, there are at least two pathways for xylitol production in Z. mobilis based on the in vitro experiments, but the extent to which they contribute to xylitol formation under physiological conditions remains to be determined.
For high-level production of ethanol, Z. mobilis is grown in high concentrations of a fermentable carbon source, which can result in osmotic shock. Osmotic shock manifests itself as a long lag period before growth commences when wild type strains are transferred to liquid media that contains >200 g/L of glucose or fructose or >360 g/L of sucrose (Loos et al. (1994) J Bacteriol 176:7688-7693). Furthermore, addition of sorbitol to the growth medium reduces the lag period when wild type strains are shifted to high concentrations of these sugars (Wiegert et al. (1996) Arch Microbiol 166:32-41, Loos et al supra).
It has also been shown that the periplasmic enzyme glucose-fructose oxidoreductase (GFOR) plays an important role in osmotic balance when wild type Z. mobilis is grown in concentrated mixtures of glucose and fructose (Loos et al. supra) or concentrated solutions of sucrose (Wiegert et al. supra, Loos et al. supra). Briefly, GFOR with its tightly bound co-factor, catalyzes the oxidation of glucose to gluconolactone and subsequent reduction of fructose to sorbitol in a classical Ping Pong Bi mechanism as shown in Diagram I. The sorbitol that is generated in the periplasmic space is transported into cells against a concentration gradient where it accumulates to high levels since it is not further metabolized. The high concentration of sorbitol inside the cells eliminates the osmotic pressure difference across the plasma membrane and restores osmotic balance.
A spontaneous mutant of wild type Z. mobilis that cannot generate sorbitol was show to produce higher levels of ethanol than wild type cells when it was grown on low concentrations of sucrose (<150 g/L), but this strain could not grow on high concentrations of sucrose (Kirk and Doelle (1993) Biotechnol. Letters 15:985-990). This mutant was subsequently shown to lack expression of glucose-fructose oxidoreductase (GFOR), which accounts for its inability to convert any of the sucrose-derived fructose to the unwanted by-product sorbitol (Wiegert et al. supra). It was also shown that growth of the sorbitol-deficient mutant in high concentrations of sucrose could be restored by adding sorbitol to the growth medium (Wiegert et al., supra). Thus GFOR plays a critical role in osmotic balance by synthesizing sorbitol when Z. mobilis is grown in concentrated mixtures of glucose and fructose or high concentrations of sucrose, which is hydrolyzed to glucose and fructose by the host cell's invertase.

CN1600850(A) discloses a non-xylose utilizing mutant strain of Z. mobilis that has an inactivated GFOR gene, and production of ethanol using this strain. The lack of sorbitol production with this strain resulted in higher levels of ethanol when glucose, fructose or sucrose was the carbon source.
The effects of reducing or eliminating glucose-fructose oxidoreductase enzyme activity in an engineered xylose-utilizing strain of Z. mobilis that is grown on a mixture of xylose and glucose (in the absence of any added sucrose or fructose) are not known.
The problem to be addressed is how to produce increased amounts of ethanol in fermentations using a xylose-utilizing Z. mobilis strain grown on xylose-containing medium. Applicants have solved the problem by determining the principle pathway for production of xylitol in vivo, and eliminating the enzyme activity that is responsible for its formation through gene inactivation, thereby creating a Z. mobilis strain that may be used for improved ethanol production.